Biostasis Pluralism

A Unified Framework

Summary

Metabolic arrest of the brain could permit the treatment, rejuvenation, and revival of people considered dead by contemporary criteria. There are currently two approaches to achieve metabolic arrest: ultra-low temperatures and chemical fixation. The use of ultra-low temperatures entails the partial replacement of the liquid parts of tissues and cells with a cryoprotectant to prevent ice formation. Chemical preservation entails a process of (aldehyde) crosslinking with the aim of structural preservation. Both types of preservation are non-reversible with contemporary technologies and will require equivalent advanced technology for revival. We propose a unified framework with standards of evidence for low temperature preservation and chemical preservation. We assert that any given revival mechanism theorized to be effective for traditional cryopreservation will also be effective in chemopreservation. We also assert that chemopreservation does not exclusively require mind uploading and cryopreservation does not exclusively entail biological revival.

Types of preservation

Metabolic arrest procedures seek to stop post-mortem ischemia and autolysis and preserve the structure of the brain, and often the entire body, to allow for potential future revival. Two core methods are commonly practiced: cryopreservation and chemopreservation.

Cryopreservation: In cryopreservation, metabolic arrest is achieved by cooling the brain to a temperature where all chemistry stops. To prevent ice formation, this entails perfusion of high concentrations of cryoprotectants (CPAs). As practiced today, it causes irreversible damage to brain tissue via osmotic injury and cryoprotectant toxicity.

Chemopreservation: In chemical fixation, aldehydes (formaldehyde and/or glutaraldehyde) are used to cross-link the molecules. Once they are cross-linked, metabolism stops. Afterwards, cryoprotectants can be slowly introduced for more stable long-term storage temperatures. As practiced today, it causes currently irreversible structural modifications to brain cells via cross-linking of proteins.

Types of revival

The shared goal of cryo- and chemopreservation is the eventual revival of the patient. While no method of reversal has been demonstrated to date on any preserved person, biostasis advocates commonly discuss a number of alternative revival methods. We classify these proposed methods into three broad families: simple revival, scan and recreate, and nanoscale repair.

Simple revival

In deep hypothermic circulatory arrest (DHCA), a patient is cooled until circulatory arrest and a flat EEG has been obtained. After 30 to 40 minutes the surgery is complete and the patient is carefully rewarmed; heart and brain activity return, and the patient generally returns to a normal, functioning state. A simple revival from human preservation might work similarly: the cryoprotectants would be unloaded from the patient’s circulatory system, the body rewarmed, and perhaps minor surgical interventions would be performed. This would constitute true human suspended animation. While obviously desirable, this type of simple revival is currently incompatible with all known preservation techniques.

Scan and recreate

Advocates of scan-and-recreate revival argue that a preserved person’s brain could eventually be scanned, either destructively or nondestructively, using advanced versions of current imaging techniques or entirely new future methods. The resulting information could be recreated in a high-fidelity computer simulation, which would then exhibit the preserved person’s memory, personality, and skills. Alternatively, future biological printers could produce a new physical form with characteristics identical to that of the preserved person, omitting damage from their cause of death and structural changes caused by the preservation itself.

Nanoscale repair

Future technology might include advanced, programmable nanomachinery, capable of interrogating individual molecules, cells, and organs of a preserved person. These nanomachines might pass through the entire body and brain, repairing and replacing damaged or altered tissue in situ. At the end of this process, the preserved person might be left in a state suitable for simple revival. Such nanoscale technology is a necessary prerequisite for biological revival of chemo- or cryopreserved people, because both methods as currently practiced cause extensive nanoscale alterations throughout the person’s body and brain.

Structural changes caused by each kind of preservation

Both cryopreservation and chemopreservation cause systematic alterations to the fine structure of the brain in the process of preserving it. Each technology displays its own characteristic set of changes, neither of which is reversible with the medical technology of today, and neither of which is compatible with the ordinary functioning of the organ.

Cryopreservation

Structural changes associated with cryopreservation fall into four major categories: cryoprotectant toxicity, osmotic damage, ice crystal formation, and fracturing.

At vitrifiable concentrations, cryoprotectants displace water molecules, also affecting water molecules in the hydration shell of the proteins which make up the human body. This can alter both the form of the protein and its interactions with its environment and cannot simply be reversed by re-hydrating the cells. Cryobiologists optimize their cryoprotectant mixtures to minimize these hydration shell perturbation effects and other forms of cryoprotectant toxicity associated with specific compounds, but it’s not possible to eliminate cryoprotectant toxicity entirely.

These same high concentrations of cryoprotectants also produce osmotic damage throughout the brain and body. This produces ultrastructural distortion and dehydration. Poor penetration of cryoprotection through the blood-brain barrier (compared to water) produces shrinking of the brain. 21st Century Medicine reports:

The water content declines from perhaps 80% to perhaps 25-30% or, expressed differently, the volume within cells, axons, and dendrites, probably declines by around 70%. This means all proteins and the like are concentrated by about three-fold. (source)

This shrinkage distorts the ultrastructure of the brain, rendering it difficult to recognize under electron microscopy in its preserved state, and precludes full restoration of viability after whole-brain cryopreservation as measured by viability assays like K/Na.

A third source of structural alteration is ice crystal formation. Cryopreservation without cryoprotection produces massive ice formation in the brain. This ice formation can be reduced by using cryoprotectants like glycerol, DMSO, or more modern multi-CPA cocktails. Cryoprotectant solutions can be formulated to inhibit ice formation completely but at the cost of producing—as discussed above—cryoprotectant toxicity. In non-ideal circumstances with post-mortem delays, incomplete penetration of the vitrification solution will produce areas that freeze instead of vitrifying. There are also non-vascularized liquid areas in the body (like the cornea) that will freeze because cryoprotectants cannot penetrate them. It is generally agreed that in almost all real-world human cryopreservation cases there is some degree of ice formation.

Finally, cold-temperature stress fracturing occurs when different parts of an organ shrink or distort at different rates, causing mechanical distortion and internal tensions. Below the glass transition temperature (in biostasis practice, below -120 °C), these tensions cause shearing and cracking throughout the brain. In areas that are frozen without cryoprotectant, fracturing will already occur at high sub-zero temperatures. Fractures can range from macro cracks in the rest of the body that can be repaired with contemporary surgical techniques to finer cracks in the brain that would require nanomedical repair. This type of fracturing can be reduced by storing just below the glass transition temperature, but these intermediate-temperature storage technologies are not available for whole-body biostasis at the time of writing.

Chemopreservation

Structural changes caused by chemopreservation fall into three major categories: aldehyde crosslinks, osmotic disruption, and long-term molecular change.

In chemical preservation, aldehydes are perfused through the vascular system, where they permeate cells and become irreversibly attached to the proteins of the body. These covalent aldehyde crosslinks stabilize the tissue and prevent autolysis. This structural change occurs throughout a preserved brain; while it preserves the form of the brain’s ultrastructure, it stops all normal metabolic processes. Current chemical techniques can sever individual aldehyde crosslinks, but cannot systematically reverse them throughout the brain and restore viability.

Because fixed tissue is highly resilient, and cryoprotectants can be introduced very slowly after initial fixation, even to very high concentrations, osmotic disruption can be limited. Shrinking observed in animal brains preserved this way is very small. The ultrastructure remains clear under electron microscopy. But compared to the gold standard of cryofixation (the use of ultra-rapid cooling of small samples), some structural changes are produced.

When done correctly, chemopreservation cases show significantly less osmotic disruption than in cryopreservation cases even when the same or higher concentration of cryoprotectant is used. However, the challenges associated with sub-zero storage remain the same: unless a sufficient concentration of cryoprotectant is introduced, and adequate perfusion allows for delivery of the cryoprotectant to every part of the brain, ice formation will also occur in a fixed brain at low temperatures.

A third potential form of structural change may occur during long-term storage of aldehyde-fixed tissues. Aldehyde fixatives are not universal cross-linkers, leaving certain biomolecules susceptible to long-term molecular change. Only a relatively small portion of biomolecules with reactive (amide) groups are actually crosslinked. While these biomolecules (such as neutral lipids) may be passively trapped or anchored within cross-linked structures, this mode of preservation is qualitatively different from direct covalent cross-linking. This consideration highlights the importance of optimizing storage temperatures for aldehyde-fixed tissues. Sub-zero temperatures combined with cryoprotectants or vitrification can be employed to eliminate long-term degradation of these non-crosslinked fractions—an active area of investigation within chemopreservation.

No biopreservation method is reversible by contemporary technologies

The framework of simple revival (suspended animation) is appealing to many people pursuing human preservation. While cryogenic preservation is simply reversible in sperm, embryos, and some small animal organs, it does not scale to larger volumes yet. The physics-based constraints of cooling and re-warming across a large volume are challenging. No organ larger than a rabbit kidney has successfully undergone the process of cryopreservation and successful transplantation with long-term survival.

If someone attempted to revive a traditionally cryopreserved person with contemporary technologies – even if that person had no underlying terminal illness – that process would not yield a remotely viable state. First there would be freezing or devitrification during the rewarming process. Ice crystals which nucleated during the cooling process would grow rapidly as the body is rewarmed. Next, whole organs, having suffered low-temperature fractures, would not be able to resume function. The fine structure of the brain would not be able to resume function without major defects (because of cryoprotectant toxicity combined with osmotic damage); if some isolated cells survived, they would soon die (either necrotically or apoptotically). The person would not regain full-body and cerebral functionality, nor could any current medical treatment restore it to them.

Similarly, there is no known method to chemically break aldehyde crosslinks inside cells. The cells are unable to function or metabolize. In other words, there is no known contemporary technology that would allow for simple revival of a chemopreserved person.

Across both cases, the story is simple. Biostasis procedures as practiced today are incompatible with simple revival. Moreover, it seems unlikely that any form of reversible human whole-body preservation will be developed in the near future.

If something is not reversible with contemporary technologies, then we are in an information-theoretic evaluation framework

Both forms of preservation available to us, then, cannot secure uniform preservation of viability. Regardless of our choice of preservation method, we must anticipate that the future will develop technology much more advanced than simple revival. Our priority then shifts to preserving identity-critical information which the future will need in order to avail ourselves of that technology. Biostasis advocates, therefore, must approach their work from the perspective of information theory.

Of course, it is difficult to predict exactly what information the future may or may not need. As we learn more about the brain, it may be possible to infer some kinds of lost information while others may be lost forever. For instance, some structures might be identical across different people such that they can be repaired or recreated based on some general model of human neurobiology; other structures might be redundant with person-specific information successfully preserved elsewhere in the same brain. There might also be a genetic component that permits broader psychological traits to be inferred without consulting the preserved structure of the brain.

Given this uncertainty, our safest path is to optimize for preserving as much structural information as possible. Preserving redundant information does no harm, whereas failing to preserve crucial information threatens the integrity of the entire preservation. The less inference the future is forced to attempt, the better we have succeeded at preservation. It is on this criterion that we must evaluate our preservation methods.

Either traditional cryopreservation or aldehyde fixation could be scanned and recreated

As described above, one revival technology is scanning and recreation. In one possible implementation of this method, a preserved brain would first be sliced with a fine tool like a microtome. The resulting slices would be imaged – perhaps after an expansion process, to allow for the use of light microscopy – and comprehensively scanned. Neurons would be traced out between slices, mapping their connections, and classified by type. A similar process could even be applied to the entire body, if that were desirable.

This method has real precedent in existing science. Scanning and tracing are possible at our current technology levels, as are very simple emulations of the brains of small organisms. As science improves and computation becomes cheaper, it may become possible to simulate a brain at high enough fidelity to qualify as a medical upload. Alternatively, we may develop methods for recreating preserved people in vivo in sub-cellular detail, reproducing the details of their brain and body exactly in a biological state. Those methods could selectively omit alterations caused by the preservation process, as well as damage from age or illness and otherwise faithfully recreate the original person.

Such a method could be compatible with traditional cryopreservation, albeit with some caveats. The imaging methods available today are designed for use on fixed tissue. In order to image cryopreserved brains, we must either develop novel imaging methods, or would have to fix the brains before imaging them—if we must do that, then it seems more sensible to chemopreserve in the first place. Another option is the use of molecular nanotechnology to image the damaged state at cryogenic temperatures as articulated in Robert Freitas’s book Cryostasis Revival.

For chemopreservation, scan-and-recreate is an easy and appealing option. Chemopreservation methods are based on exactly the kind of standard tissue preparation methods used on samples intended for electron microscopy. A chemopreserved brain is amenable to washout, expansion, slicing, and imaging. End-to-end tests validate that chemopreservation preserves traceable synapses and even subcellular structures. If chemopreservation is combined with low-temperature storage or even vitrification, this method will only work if no ice is formed during cooling or devitrification happens during the warming process.

In either chemopreservation or cryopreservation, it may become possible to nondestructively scan whole volumes of organs. While this is not possible today, a nondestructive scan would enable both uploading and biological recreation of an individual without destroying the original preserved patient.

Either traditional cryopreservation or aldehyde fixation could be repaired by molecular nanotechnology

The other revival technology might be the use of advanced nanomachinery. Very small programmed robots could pass through the preserved body, performing nanoscale alterations to reverse changes caused by the preservation process. These nanobots might reverse other kinds of damage at the same time, undoing the undesirable effects of age or illness. As with scanning and recreation, the preserved structural information is key to this process: the machines must be able to deduce what healthy state they should restore the preserved person to.

The implementation details of this kind of nanomachine repair might vary. In the most straightforward kind of process, the machines might enter the cells, at lower temperatures first, directly repairing damaged molecules one by one and transporting out waste products. Nanomachines working with chemopreserved tissue would have an easy time breaking the aldehyde crosslinks; current technology can already sever various types of crosslinks, and nanomachines could target solutions to the specific crosslink as needed. In a traditional cryopreservation, or in a chemopreservation that included subzero temperatures, more inference might be necessary if ice crystals and stress fractures have obscured the original structure.

Alternatively, nanomachines need not save the existing cellular structures. A different design might travel through the body cell-by-cell, destructively reading the contents of each individual cell before printing a healthy replacement. People of the future might consider this process analogous to the normal regenerative processes of the body, through which cells constantly die and are replaced. Again, this process would be compatible either with chemopreservation (where the nanomachines could print aldehyde-free replacement cells) or traditional cryopreservation.

Evidence Standards for Both Kinds of Preservation

To evaluate the quality of a given type of preservation in an information-theoretic context, whether cryopreservation or chemopreservation, the evaluation criteria are effectively the same for any given preservation method. Each process must be evaluated on the basis of whether it preserves the neuroanatomical basis of a specific individual.

To do this, a preservation method must preserve proteins, DNA, differentiating cellular features, intercellular relationships, and in particular the synapses—the full connectomic map of synaptic connections throughout the entire brain. Confirming the presence and adequate preservation of these features can only feasibly be done with a combination of large-scale imaging and spot-checking individual regions with nanoscale imaging across as many regions of the entire brain as possible. Even very badly preserved brains may look perfect in nanoscale imaging of a few carefully selected regions, and many lower resolution imaging techniques like light microscopy may look perfect even when nanoscale imaging shows the underlying ultrastructure is destroyed.

Traditional cryopreservation techniques historically have aimed for preservation of viability, but advocates of cryopreservation are not exempt from presenting compelling evidence of preservation of identity-critical structure because cellular viability assays (even if applied to an organ) can co-exist with significant (regional) osmotic and structural damage.

Ideal and non-ideal cases

Neither chemopreservation nor traditional cryopreservation is compatible with simple revival. With that option removed from consideration, the most promising remaining options – scan-and-recreate or nanorepair – are both constrained chiefly by the quality of the information preserved in the neural structure. Electron microscopy shows that high-quality aldehyde fixation preserves this information in excellent detail. Traditional brain cryopreservation under optimal conditions is still currently characterized by ultrastructural signatures of osmotic cell damage and CPA-induced brain shrinking.

Cryopreservation and aldehyde fixation impose special constraints on the post-mortem interval for perfusion. In either case, perfusion must begin within minutes after clinical death to avoid the “no-reflow” phenomenon. Where delays are longer than about fifteen minutes, some areas receive compromised flow, and fixation and cryoprotection are both compromised. Carrier solution composition and perfusion protocols may be able to extend this period, but for periods of extensive ischemia, perfusion impairment must be assumed. For both cryopreservation and chemopreservation, this means accepting that some areas of the brain may have low levels of cryoprotectant or fixative and will therefore become damaged by ice or decay.

It is currently an open question whether freezing (cryopreservation without cryoprotection) or immersion of the brain in chemical fixative (possibly with subsequent immersion in cryoprotectant) yields the best structural preservation for such non-ideal cases. Currently, our position is that a strong evidence-based case is lacking for how to optimize structural preservation for cases where perfusion would not yield good distribution of any preservative agent. It is even possible that for cases with moderate ischemia, immersion chemical fixation would be favored, and for cases with very extensive ischemia, straight freezing would be favored. The framework that we present in this article cannot conclusively answer these questions without conducting more empirical research.

Conclusion

Cryopreservation and chemopreservation are distinct technologies to produce metabolic arrest in patients that can no longer be treated by contemporary medicine. Both technologies produce molecular and structural changes that cannot be reversed by contemporary technologies. Simple revival (suspended animation) is not possible and either future scan-and-recrate or molecular repair technologies are needed for revival. While people can disagree about the importance of viability preservation in contemporary preservation protocols, advocates of both technologies should produce the best ultrastructural evidence possible. While chemopreservation has historically been linked to “mind uploading” and cryopreservation to biological molecular repair, both technologies are compatible with either revival technology. Cryopreservation indisputably produces metabolic arrest but today’s vitrification technologies still produce ultrastructural signs of osmotic cell damage and CPA-induced brain shrinking. Chemopreservation mostly avoids these issues but optimal and practical storage temperatures still require definitive resolution.

Under non-ideal conditions it is not clear which technology is favored for optimal ultrastructural preservation given the trade-off between cellular decomposition in immersion fixation and ice formation when the brain is frozen without cryoprotectant.